Neuraminidase Gene Variations in Influenza A(H1N1)pdm09 Virus among Patients Admitted to Refferal Pulmonary Hospital, Tehran, Iran in 2009-2013.

Background
Neuraminidase (NA) is one of the surface proteins of influenza A virus, which plays an important role in immunization against influenza infection and is recognized as an important therapeutic target. Genetic and antigenic changes and substitutions can influence the efficacy of vaccine and change viral sensitivity to NA inhibitors (NAIs). In this study, we performed phylogenetic and molecular analyses of NA changes in influenza A(H1N1)pdm09 virus, compared them with the corresponding vaccine strain, and examined drug resistance mutations in isolates from patients.


Materials and Methods
The complete sequence of NA genes from 34 pandemic H1N1 isolates (identified in 2009-2010, 2010-2011, and 2013) was determined and analyzed both genetically and antigenically. The phylogenetic tree was plotted relative to the corresponding vaccine strain, using MEGA6 software package, based on the maximum likelihood method and JTT matrix (bootstrap value of 1000).


Results
The phylogenetic analysis of pandemic isolates showed 31 amino acid substitutions in NA genes, compared to the vaccine strain. Some of these substitutions (N248D, V241I, N369K, N44S, and N200S) were important in terms of phylogenetic relationship, while the rest (D103N, V106I, R130T, N200S, G201E, and G414R) influenced the antigenic indices of B-cell epitopes. The catalytic sites, framework sites, and N-glycosylation remained unchanged in the studied samples. Meanwhile, H275Y substitution, related to oseltamivir resistance, was detected in 3 isolates. The average nucleotide identity of NAs with the corresponding vaccine strain was 99.415%, 98.607%, and 98.075% in 2009-2010, 2010-2011, and 2012-2013, respectively.


Conclusion
In this study, we provided basic information on the genetic and antigenic changes of NA genes in influenza A(H1N1)pdm09 virus from patients in 3 different seasons in Tehran, Iran. Considering the viral NAI resistance and changes in NA gene sequences of the isolates in comparison with the vaccine strain, further studies should be performed to monitor genetic changes in Iran. Moreover, the efficacy of vaccines should be examined.


INTRODUCTION
Influenza viruses are associated with significant morbidity and mortality and affect approximately 20% of the world's population each year (1). In general, vaccination can provide optimal protection against influenza viruses. However, the level of protection by annual vaccination can be limited due to antigenic adaptation of circulating strains. In addition, the low level of vaccine coverage is another major issue in different communities (2).
Besides immunization, use of antiviral medicines, along with therapeutic or preventive strategies, can help prevent morbidity, mortality, and spread of influenza viruses (3). In addition, NA is involved in the releasing of progeny virions, viral cell-to-cell spread, HA-mediated fusion, and effective virus proliferation (8)(9)(10).
Over the past decades, NA has been considered as a major drug target. Today, NA inhibitors (NAIs), such as oseltamivir, zanamivir, inhaled laninamivir, and peramivir, are available and used against influenza viruses, including A(H1N1)pdm09 virus (11). On the other hand, resistance mutations (H275Y, E119V/G, and I22V) have been reported in in vivo and in vitro models (12).
Since the emergence of influenza A(H1N1)pdm09 virus (13), many studies around the world have evaluated its genetic properties, drug resistance patterns, and virulent mutations. The Iranian population has been affected by the pandemic influenza virus since 2009. In our previous studies, we discussed the clinical features of influenza A(H1N1)pdm09 virus (14)(15)(16). In addition, we performed a study on the prevalence of oseltamivir resistance and H275Y mutations in pandemic H1N1 strains during 2009-2010 season, using real-time probe-based polymerase chain reaction (PCR) method (17).
With this background in mind, in the present study, we aimed to perform molecular and phylogenetic analyses of NA genes from 2009 pandemic H1N1 influenza viruses (identified in 2009-2010, 2010-2011, and 2012-2013) in patients referring to Masih Daneshvari Hospital, a specialized and referral center for lung diseases in Tehran, Iran. We also identified the genetic and antigenic changes with respect to the vaccine strain and monitored NAI resistance mutations.

Samples
In this retrospective, cross sectional study, 34

Phylogenetic analysis
The PCR products of NA genes were sequenced, using       (Table 3). Table 4 presents the site and sequence of N-linked glycosylation in the studied samples. All 8 cases of Nlinked glycolisation remained unchanged in the NA genes of pandemic strains (Table 4). In addition,    Bootstrap support values after 1000 pseudo-random replicates higher than 50% are shown in the corresponding nodes.   (Table 2). In addition, Table 3 presents a sequential analysis of changes in NA genes relative to the vaccine strain at amino acid level.
Based on the present findings, although catalytic, framework, and glycosylation sites did not change in NA genes, 31 amino acid substitutions were detected in other regions. Overall, N44S, V62I, V106I, N200S, V241I, N248D, and N369K substitutions were most commonly detected (Table 3). Some other mutations have been also reported in other studies in Iran (24), which are of phylogenic importance.
The phylogenetic tree shows that the strains studied in each season constitute separate branches in the tree ( Figure   1)  Meanwhile, H275Y substitution, detected in this study, was of phylogenic importance and led to speciation of influenza strains in the phylogenetic tree ( Figure 1). As stated earlier, in addition to enzymatic activity, NA exhibits antigenic properties. Therefore, in the study of influenza A(H1N1)pdm09 virus, antigenic changes in NA protein should be also investigated.
In the present study, examination of mutations in the epitopic regions with respect to the vaccine strain revealed changes in reserved B-cell-dependent antigenic parameters (Table 5)